Chemiluminescent 1,2-dioxetane compounds

ABSTRACT

Novel light producing 1,2-dioxetanes are described of the formula ##STR1## wherein ArOX is an aryl ring substituted with an X oxy group and A are passive organic groups which allow the 1,2-dioxetane to produce light when triggered by removing X. X is a chemically labile group which is removed by an activating agent. The 1,2-dioxetane compounds can be triggered to produce light at room temperatures.

This application is a division of application Ser. No. 887,139, filedJul. 17, 1986.

BACKGROUND OF THE INVENTION

(1) Statement of the Invention

The present invention relates to chemiluminescent 1,2-dioxetanecompounds which can be triggered by an activating agent to generatelight. In particular, the present invention relates to stable aryl groupsubstituted 1,2-dioxetanes which contain an activatable oxide group (OX)which is ring substituted in the aryl group, wherein the stable1,2-dioxetane forms an unstable 1,2-dioxetane compound by removal of Xwhich decomposes to light and two carbonyl containing compounds.

(2) Prior Art

1. Mechanisms of Luminescence. Exothermic chemical reactions releaseenergy during the course of the reaction. In virtually all cases, thisenergy is in the form of vibrational excitation or heat. However, a fewchemical processes generate light or chemiluminescence instead of heat.The mechanism for light production involves two steps: (1) thermal orcatalyzed decomposition of a high energy material (generally a peroxide)yields one of the reaction products in a triplet or singlet electronicexcited state and (2) emission of a photon (fluorescence orphosphorescence) from this excited species produces the light observedfrom the reaction. ##STR2##

2. Dioxetane Intermediates in Bioluminescence. In 1968 McCapra proposedthat 1,2-dioxetanes might be the key high-energy intermediates invarious bioluminescent reactions including the firefly system. (F.McCapra. Chem. Commun., 155 (1968)). Although this unstable dioxetaneintermediate has not been isolated nor observed spectroscopically,unambiguous evidence for its intermediacy in this biochemical reactionhas been provided by oxygen-18 labelling experiments. (O. Shimomura andF. H. Johnson, Photochem. Photobiol., 30, 89 (1979)). ##STR3##

3. First Synthesis of Authentic 1,2-Dioxetanes. In 1969 Kopecky andMumford reported the first synthesis of a dioxetane(3,3,4-trimethyl-1,2-dioxetane) by the base-catalyzed cyclization of abeta-bromohydroperoxide. (K. R. Kopecky and C. Mumford, Can. J. Chem.,47,709 (1969)). As predicted by McCapra, this dioxetane did, in fact,produce chemiluminescence upon heating to 50° C. with decomposition toacetone and acetaldehyde. However, this peroxide is relatively unstableand cannot be stored at room temperature (25° C.) without rapiddecomposition. In addition, the chemiluminescence efficiency is very low(less than 0.1%). This inefficiency is due to two factors: (1) thebiradical nature of the mechanism for its decomposition and (2) the lowquantum yield of fluorescence of the carbonyl cleavage products.##STR4##

Bartlett and Schaap and Mazur and Foote independently developed analternate and more convenient synthetic route to 1,2-dioxetanes.Photooxygenation of properly-substituted alkenes in the presence ofmolecular oxygen and a photosensitizing dye produces the dioxetanes inhigh yields. (P. D. Bartlett and A. P. Schaap, J. Amer. Chem. Soc., 92,3223 (1970) and S. Mazur and C. S. Foote, J. Amer. Chem. Soc., 92 3225(1970)). The mechanism of this reaction involves the photochemicalgeneration of a metastable species known as singlet oxygen whichundergoes 2+2 cycloaddition with the alkene to give the dioxetane.Research has shown that a variety of dioxetanes can be prepared usingthis reaction (A. P. Schaap, P. A. Burns, and K. A. Zaklika, J. Amer.Chem. Soc., 99, 1270 (1977); K. A. Zaklika, P. A. Burns, and A. P.Schaap, J. Amer. Chem. Soc., 100, 318 (1978); K. A. Zaklika, A. L.Thayer, and A. P. Schaap, J. Amer. Chem. Soc., 100, 4916 (1978); K. A.Zaklika, T. Kissel, A. L. Thayer, P. A. Burns, and A. P. Schaap,Photochem. Photobiol., 30, 35 (1979); and A. P. Schaap, A. L. Thayer,and K. Kees, Organic Photochemical Synthesis II, 49 (1976)). During thecourse of this research, a polymer-bound sensitizer for ##STR5##photooxygenations was developed (A. P. Schaap, A. L. Thayer, E. C.Blossey, and D.C. Neckers, J. Amer. Chem. Soc., 97, 3741 (1975); and A.P. Schaap, A. L. Thayer, K. A. Zaklika, and P. C. Valenti, J. Amer.Chem. Soc., 101, 4016 (1979)). This new type of sensitizer has beenpatented and sold under the tradename SENSITOX™ (U.S. Pat. No. 4,315,998(Feb. 16, 1982); Canadian Patent No. 1,044,639 (Dec. 19, 1979)). Overfifty references have appeared in the literature reporting the use ofthis product.

4. Preparation of Stable dioxetanes Derived from Sterically HinderedAlkenes. Wynberg discovered that photooxygenation of sterically hinderedalkenes such as adamantylideneadamantane affords a very stable dioxetane(J. H. Wieringa, J. Strating, H. Wynberg, and W. Adam, TetrahedronLett., 169 (1972)). A collaborative study by Turro and Schaap showedthat this dioxetane exhibits an activation energy for decomposition of37 kcal/mol and a half-life at room ##STR6## temperature (25° C.) ofover 20 years (N. J. Turro, G. Schuster, H. C. Steinmetzer, G. R. Falerand A. P. Schaap, J. Amer. Chem. Soc., 97, 7110 (1975)). In fact, thisis the most stable dioxetane yet reported in the literature. Adam andWynberg have recently suggested that functionalizedadamantylideneadamantane 1,2-dioxetanes may be useful for biomedicalapplications (W. Adam, C. Babatsikos, and G. Cilento, Z. Naturforsch.,39b, 679 (1984); H. Wynberg, E. W. Meijer, and J. C. Hummelen, InBioluminescence and Chemiluminescence, M. A. DeLuca and W. D. McElroy(Eds.). Academic Press, New York, p. 687, 1981). However, use of thisextraordinarily stable peroxide for chemiluminescent labels wouldrequire detection temperatures of 150° to 250° C. Clearly, theseconditions are unsuitable for the evaluation of biological analytes inaqueous media. Further, the products (adamantanones) of these dioxetanesare only weakly fluorescent so that the chemiluminescent decompositionof these proposed immunoassay labels is very inefficient. McCapra, Adam,and Foote have shown that incorporation of a spirofused cyclic orpolycyclic alkyl group with a dioxetane can help to stabilize dioxetanesthat are relatively unstable in the absence of this sterically bulkygroup (F. McCapra, I. Beheshti, A. Burford, R. A. Harm, and K. A.Zaklika, J. Chem. Soc., Chem. Commun., 944 (1977); W. Adam, L. A. A.Encarnacion, and K. Zinner, Chem. Ber., 116, 839 (1983); and G. G.Geller, C. S. Foote, and D. B. Pechman, Tetrahedron Lett., 673 (1983)).

5. Effects of Substituents on Dioxetane Chemiluminescence. The stabilityand the chemiluminescence efficiency of dioxetanes can be altered by theattachment of specific substituents to the peroxide ring (K. A. Zaklika,T. Kissel, A. L. Thayer, P. A. Burns, and A. P. Schaap, Photochem.Photobiol., 30, 35 (1979); A. P. Schaap and S. Gagnon, J. Amer. Chem.Soc., 104, 3504 (1982); A. P. Schaap, S. Gagnon, and K. A. Zaklika,Tetrahedron Lett., 2943 (1982); and R. S. Handley, A. J. Stern, and A.P. Schaap, Tetrahedron Lett., 3183 (1985)). The results with thebicyclic system shown below illustrate the profound effect of variousfunctional groups on the properties of dioxetanes. Thehydroxy-substituted dioxetane (X═OH) derived from the2,3-diaryl-1,4-dioxene exhibits a half-life for decomposition at roomtemperature ##STR7## (25° C.) of 57 hours and produces very low levelsof luminescence upon heating at elevated temperatures. In contrast,however, reaction of this dioxetane with a base at -30/° C. affords aflash of blue light. Kinetic studies have shown that the deprotonateddioxetane (X═O⁻) decomposes 5.7×10⁶ times faster than the protonatedform (X═OH) at 25° C.

The differences in the properties of these two dioxetanes arise becauseof two competing mechanisms for decomposition (K. A. Zaklika, T. Kissel,A. L. Thayer, P. A. Burns, and A. P. Schaap, Photochem. Photobiol., 30,35 (1979); A. P. Schaap and S. Gagnon, J. Amer. Chem. Soc., 104 3504(1982); A. P. Schaap, S. Gagnon, and K. A. Zaklika, Tetrahedron Lett.,2943 (1982); and R. S. Handley, A. J. Stern, and A. P. Schaap,Tetrahedron Lett., 3183 (1985). Stable dioxetanes cleave by a processthat requires approximately 25 kcal for homolysis of the O--O bond andformation of a biradical. An alternative mechanism for decomposition isavailable to dioxetanes bearing substituents such as O⁻ with lowoxidation potentials. The cleavage is initiated by intramolecularelectron transfer from the substituent to the antibonding orbital of theperoxide bond. In contrast to the biradical mechanism, theelectron-transfer process generates chemiluminescence with highefficiency.

Literature Examples Related to Triggering of dioxetanes

(a) Base Triggering of Dioxetanes. The only example in the literature isdescribed above (A. P. Schaap and S. Gagnon, J. Amer. Chem. Soc., 104,3504 (1982). The hydroxy-substituted dioxetane shown above is toounstable to be of use in any application. It has a half-life at 25° C.of only 57 hours. Neither the dioxetane nor the precursor alkene wouldsurvive the conditions necessary to prepare derivatives.

(b) Fluoride Triggering of dioxetanes. No examples appear in theliterature with dioxetanes. Fluoride is used synthetically to desilylatealcohol derivatives. (E. J. Corey and A. Venkateswarlu, J. Amer. Chem.Soc., 94, 6190 (1972).

(c) Enzymatic Triggering of Dioxetanes. No examples appear in theliterature with dioxetanes. Enzymes have been used in ColorimetricImmunoassays and Fluorometric Immunoassays to remove phosphate,beta-D-galactoside, and other groups with resulting color development orformation of fluorescent materials (L. J. Kricka, In Ligand-BinderAssays, Marcel Dekker, Inc., New York, p. 170 (1985). There are numerousexamples of chemiluminescence immunoassays (L. J. Kricka, InLigand-Binder Assays, Marcel Dekker, Inc., New York, p. 199 (1985)) butno cases with a stable dioxetane that is triggered.

(d) Japanese Patent Application 57042686 filed Mar. 10, 1982 and FrenchPatent No. 2,383,404 describe various unrelated dioxetanes. U.S. Pat.No. 3,720,622 describes unrelated light producing compounds.

OBJECTS

It is therefore an object of the present invention to provide novelstable 1,2-dioxetanes which can be decomposed with an activating agentto form light and two carbonyl compounds. Further it is an object of thepresent invention to provide 1,2-dioxetanes which are stable at roomtemperatures over an extended period of time. Further still it is anobject of the present invention to provide 1,2-dioxetanes which areactivatable by chemical and by biochemical means. Further still it is anobject of the present invention to provide a method for the use of thestable 1,2-dioxetanes to generate light. These and other objects willbecome increasingly apparent by reference to the following descriptionand the drawings.

IN THE DRAWINGS

FIG. 1 is a graph showing light intensity as a function of wavelengthfor compound 2c described hereinafter and one of its carbonyl containingcompound decomposition products, where the activating agent is afluoride.

FIG. 2 is a graph showing light intensity as a function of wavelengthfor compound 2e described hereinafter, and one of its carbonylcontaining compound decomposition products, where the activating agentis an enzyme.

The present invention relates to a stable 1,2-dioxetane compound of theformula ##STR8## wherein ArOX an aryl group having an aryl ringsubstituted with an X-oxy group which forms an unstable oxideintermediate 1,2-dioxetane compound when triggered to remove X by anactivating agent so that the unstable 1,2-dioxetane compound decomposesto form light and two carbonyl containing compounds of the formula##STR9## wherein X is a chemically labile group which is removed by theactivating agent to form the unstable oxide intermediate 1,2-dioxetaneand wherein A are passive organic groups which allow the light to beproduced.

In particular the present invention relates to a stable 1,2-dioxetanecompound of the formula ##STR10## wherein R₁ and R₂ together and R₃ andR₄ together can be joined as spirofused alkylene and aryl rings, whereinat least one of R₁ and R₂ or R₃ and R₄ is an aryl group, having an arylring substituted with an X oxy- group which forms an unstable oxideintermediate 1,2 dioxetane compound when triggered to remove X by anactivating agent selected from acids, bases, salts, enzymes, inorganicand organic catalysts and electron donors so that the unstable1,2-dioxetane compound decomposes to form light and two carbonylcontaining compounds of the formula: ##STR11## wherein those of R₁, R₂,R₃ or R₄ which are unsubstituted by an X-oxy group are carbon containingorganic groups which provide stability for the stable 1,2-dioxetanecompound and wherein X is a chemically labile group which is removed bythe activating agent to form the unstable oxide intermediate.

Further the present invention relates to a stable dioxetane compound ofthe formula: ##STR12## wherein R₁ is selected from alkyl, alkoxy,aryloxy, dialkyl or aryl amino, trialkyl or aryl silyloxy and arylgroups including spirofused aryl groups with R₂, wherein R₂ is an arylgroup which can include R₁ and is substituted with an X-oxy group whichforms an unstable oxide intermediate 1,2-dioxetane compound whenactivated by an activating agent to remove X selected from acids, bases,salts, enzymes, inorganic and organic catalysts and electron donors sothat the unstable 1,2-dioxetane compound decomposes to form light andtwo carbonyl containing compounds of the formula: ##STR13## wherein X isa chemically labile group which is removed by the activating agent toform the unstable oxide intermediate and wherein R₃ and R₄ are selectedfrom aryl and alkyl groups which can be joined together as spirofusedpolycyclic alkyl and polycyclic aryl groups.

Specifically the present invention relates to a stable 1,2-dioxetanecompound of the formula: ##STR14## wherein R₁ is selected from loweralkyl containing 1 to 8 carbon atoms, R₂ is selected from aryl, biaryland fused ring polycyclic aryl groups which can be substituted orunsubstituted, and ##STR15## is selected from polycyclic alkyl groupscontaining 6 to 30 carbon atoms, wherein OX is an oxy group substitutedon an aryl ring which forms an unstable oxide intermediate 1,2-dioxetanecompound when triggered to remove X by an activating agent selected fromacid, base, salt, enzyme, inorganic and organic catalysts and electrondonor sources and X is a chemically labile group which is removed by theactivating agent to form the unstable oxide intermediate and wherein (I)decomposes in the presence of an activating agent to produce light andcarbonyl containing compounds of the formula ##STR16##

Finally the present invention relates to a stable 1,2-dioxetane compoundof the formula: ##STR17## wherein ArOX is a spirofused aryl groupcontaining a ring substituted X-oxy group, wherein OX forms an unstableoxide intermediate 1,2-dioxetane compound when triggered by anactivating agent to remove X selected from acids, bases, salts, enzymes,inorganic and organic catalysts and electron donors, wherein X is achemically labile group which is removed by the activating agent to formthe unstable oxide intermediate 1,2-dioxetane so that the unstable1,2-dioxetane compound decomposes to form light and two carbonylcontaining derivatives of the formula ##STR18## and wherein ##STR19## isselected from polycyclic alkyl groups containing 6 to 30 carbon atoms.In this structure R₁ and R₂ are joined together.

When R₁ is not combined with R₂ the group is preferably alkyl, alkoxy,dialkyl or arylamino trialkyl or aryl silyloxy. The alkyl groupspreferably contain 1 to 8 carbon atoms. R₁ can also be cyclic aliphaticor aryl groups, including fused ring aryl compounds, containing 6 to 14carbon atoms. When R₁ is combined with R₂ they provide an aryl groupcontaining 6 to 30 carbon atoms.

R₂ is an aryl group substituted with an X oxy (OX) group. The arylcontaining group can be phenyl, biphenyl, fused phenyl and other arylgroups and can contain between 6 and 30 carbon atoms and can includeother substituents. X is any labile group which is removed by anactivating agent. The OX group can be for instance selected fromhydroxyl, alkyl or aryl carboxyl ester, inorganic oxy acid salt,particularly a phosphate or sulfate, alkyl or aryl silyloxy and oxygenpyranoside groups.

R₃ and R₄ can be the same as R₁. In the following Examples, R₃ and R₄are combined together to form a polycyclic alkylene group, particularlyfor ease of synthesis and comparison; however any organic group can beused. Preferably the polycyclic alkylene group contains 6 to 30 carbonatoms.

The stable 1,2-dioxetane compounds have relatively long 1/2 lives atroom temperatures (20°-35° C.) even though they can be triggered by theactivating agent. All of the prior art compounds are either unstable atroom temperatures or require temperatures of 50° C. or above in order tobe thermally decomposed which is impractical for most applications.

The activating agent may be chemical or enzymatic. In some cases (F⁻) 1equivalent is required and in others (enzymatic) only a very smallamount is used. The agents are described in any standard chemicaltreatise on the subject and include acids, bases, salts, enzymes andother inorganic, organic catalysts. The agent used will depend upon theconditions under which the stable 1,2-dioxetane is to be activated andhow labile the X group is on a particular 1,2-dioxetane. Electron donorscan be used to remove X which can include reducing agents as well aselectrical sources of electrons.

The 1,2-dioxetane decomposes to form carbonyl containing compounds andlight. An unstable 1,2-dioxetane intermediate is formed of the formula:##STR20##

In general an -ArOX substituted 1,2-dioxetanes are formed by addition ofoxygen to the appropriate alkene. These alkenes are synthesized throughan alkyl or aryl silyloxyaryl ring substituted intermediate. Thus theappropriate ketones of the formula: ##STR21## are reacted in thepresence of lithium aluminum hydride or other metal hydride in a polarorganic solvent, particularly tetrahydrofuran, with a transition metalhalide salt, particularly titanium chloride, and a tertiary amine base.The reaction is generally conducted in refluxing tetrahydrofuran andusually goes to completion in about 4 to 24 hours. 1,2-DioxetaneCompounds Synthesized ##STR22## Instrumentation

Nuclear magnetic resonance (NMR) spectra were obtained on either aNicolet NT300™ or a General Electric QE300™ spectrometer as solutions inCDCl₃ with tetramethylsilane as internal standard unless notedotherwise. Infrared (IR) spectra were obtained on either a Nicolet™ or aBeckman Acculab 8™ spectrometer. Mass spectra were obtained on either aKratos™ or an AEI MS-90™ spectrometer. Ultraviolet and visibleabsorption spectra were obtained on a Varian Cary 219™spectrophotometer. High performance liquid chromatography (HPLC) wasperformed with a Varian 5020 LC™. Fluorescence spectra were recorded oneither an Aminco-Bowman™ or a Spex Fluorolog/™ spectrophotofluorometer.Chemiluminescence spectra were measured using either the SpexFluorometer or a device constructed in this laboratory. Kineticmeasurements were made using another device built in this laboratorywhich is interfaced to an Apple IIe™ computer. Elemental analyses wereperformed by Midwest Microlabs, Indianapolis. Melting points weremeasured in a Thomas Hoover™ capillary melting apparatus and areuncorrected. Precision weights were obtained on a Cahn model 4700™electrobalance.

Materials

The solvents: o-xylene, toluene, propylene carbonate,N,N-dimethylformamide, N-methylpyrrolidinone, 2-methoxyethanol,1,2-dimethoxyethane, and nonane were obtained from Burdick and JacksonLaboratories and used as received for kinetic and spectroscopemeasurements. Methylcyclohexane was purified by passage over neutralalumina and fractional distillation. 1,4-Dioxane was distilled fromsodium and then from Na₄ EDTA. 9,10-Diphenylanthracene and9,10-dibromoanthracene were recrystallized from either o-xylene or2-methoxyethanol. Silica, alumina and the other solid supports wereobtained from various commercial sources as noted and used withoutfurther purification.

Syntheses of Alkenes

Methoxy (2-naphthyl)methylene!adamantane (1a). To a dry 250 mLthree-neck flask containing 100 mL of dry THF cooled to 0° C. was added12.5 g of a 2:1 mixture of TiCl₃ and lithium aluminum hydride in smallportions. An atmosphere of nitrogen was maintained throughout thereaction. The black mixture was warmed to room temperature and oftriethylamine (6.0 mL, 6 eq) was added. The reaction mixture wasrefluxed for two hours at which time addition of a solution of methyl2-naphthoate (1.34 g, 7.2 mmol) and adamantanone (1.08 g, 7.2 mmol) in50 mL of dry THF was begun. The addition was completed after 10 hoursand reflux was maintained for an additional 10 hours. ##STR23##

The cooled solution was quenched by slow addition of 5 mL of methanolfollowed by 10 mL of water. The cooled black mixture was diluted with150 mL of ether and filtered through filter paper. The ether solutionwas washed repeatedly with water until the water did not become colored.The ether solution was dried with MgSO₄ and evaporated to a yellow oilcontaining some solid (2-adamantanol). Column chromatography on silicagel with 2.5% ethyl acetate/hexane afforded 1.08 g of a clear oil whichcrystallized slowly on standing. Recrystallization from cold pentaneproduced 500 mg of 1a as white crystals: mp 68° C.; ¹ H NMR delta1.80-2.03 (m, 13H), 2.697 (s,1H), 3.325 (s,3H), 7.43-7.85 (m, 6H); ¹³ CNMR delta 28.39, 30.30, 32.36, 37.25, 39.12, 39.27, 57.77, 125.89,125.98, 127.42, 127.58, 128,02, 128,27, 132.02, 132.82, 133.15, 143.66;IR (KBr) 3055, 2910, 2850, 1680, 1630, 1600, 1210, 1090, 820,750 cm⁻¹ ;MS m/e (rel. intensity) 304 (100), 247 (27), 551 (40), 141 (17), 127(38), 57 (66).

1,6-Dibromo-2-naphthol. A 200 mL three-neck round bottom flask fittedwith condenser, addition funnel and gas outlet tube as charged with2-naphthol (21.6 g, 150 mmol) in 60 mL of glacial acetic acid. Asolution of bromine (48 g, 300 mmol) in 15 mL of acetic was addeddropwise. On completion the warm solution was heated on a steam bath for90 minutes. A solution of KOH in water was used to scrub the HBr evolvedthrough the outlet during the heating. On standing overnight at roomtemperature the product crystallized. The contents were cooled to 0° C.and filtered with suction. The light brown product weighed 41.5 g (92%)after air drying and was sufficiently pure for use in the next step.##STR24##

6-Bromo-2-naphthol. To a solution of 225 mL of ethanol and 90 mL ofconc. HCl in a 500 mL round bottom flask were added of tin metal (32.6g, 274 mmol) and 1,6-dibromo-2-naphthol (41.5 g, 137 mmol). The reactionmixture was refluxed on a steam bath for 9 hours. TLC (SiO₂, 15:1benzene/ethyl acetate) indicated consumption of starting material. Thecooled solution was decanted from unreacted tin, concentrated to 150-200mL on vacuo and poured into 600 mL of ice and water. The whiteprecipitate was collected on a Buchner funnel and dried in the air toafford 31.5 g of an off-white solid. Recrystallization from benzeneproduced 23.8 g of pure product (78%): mp 127°-127.5° C.; lit. mp127°-129° C.; reference: C. R. Koelsch, Org. Syn. Coll. Vol. 3, 132(1955). ##STR25##

6-Hydroxy-2-naphthoic acid. A 500 mL three-neck flask fitted withmagnetic stirrer, nitrogen lines and a 125 mL addition funnel wascharged with 200 mL of dry ether (newly opened can) and6-bromo-2-naphthol (15.6 g, 70 mmol). The atmosphere was replaced withnitrogen and a solution of 15 mL of 10M n-BuLi in 100 mL of ether (150mmol) was added via the addition funnel over a 30 minute period. Thesolution became pale yellow with a precipitate. After stirring for 20minutes more, dry ice was added until the solution became quite cold(<-25° C.) and green in color. The solution was warmed to roomtemperature and quenched by the addition of 200 mL of water. Thetwo-phase system was transferred to a separatory funnel, the layersseparated and the ether solution extracted with 100 mL of saturatedNaHCO₃ solution. The combined aqueous layers were washed with 100 mL ofether and neutralized by careful addition of 12N HCl. The pale bluesolid was filtered and dried in the air to give 10.3 g (76%): mp238-241° C. (dec.); lit. mp 240°-241° C.; reference: S. V. Sunthankarand H. Gilman, J. Org. Chem., 16, 8 (1951). ##STR26##

Methyl 6-hydroxy-2-naphthoate. 6-Hydroxy-2-naphthoic acid (5.0 g, 26.6mmol) was dissolved in 125 mL of methanol and refluxed with 6 drops ofconc. H₂ SO₄ for 36 hours. TLC analysis (SiO₂, 10:1 CHCl₃ /MeOH)revealed only a trace of the acid left. The solution was cooledpartially and concentrated to dryness on a rotary evaporator. The solidresidue was dissolved in 200 mL of ether and washed successively with100 mL of saturated aq. NaHCO₃ and brine. Drying over MgSO₄ andevaporating the solvent left 4.6 g of (85.5%) slightly yellow solidwhich showed only one spot on TLC. The material is sufficiently pure foruse in subsequent reactions but may be purified further byrecrystallization from ether affording a white solid mp 169°-169.5° C.;¹ H NMR delta 3.976 (s, 3H), 5.3 (br. s, 1H), 7.16-8.54 (m, 6H); IR(KBr) 3370, 1680, 1630, 1435, 1310, 1210 cm⁻¹. ##STR27##

Methyl 6-tert-butyldimethylsilyloxy-2-naphthoate. A 10 mL round bottomflask fitted with magnetic stirrer and pressure-equalizing droppingfunnel was charged with 3 mL of DMF which had been dried by vacuumdistillation from CaH₂. Methyl 6-hydroxy-2-naphthoate (1.01 g, 5 mmol)and t-butyldimethyl silyl chloride (0.83 g, 5.5 mmol) were added and theatmosphere replaced with nitrogen. A solution of imidazole (0.75 g, 11mmol) in 3 mL of dry DMF was added via the dropping funnel over 15minutes, and stirring continued for 4 hours. TLC analysis (SiO₂, 5%ethyl acetate/hexane) showed clean conversion to a new material. Thesolution was poured into 50 mL of 1% aq. Na₂ CO₃ solution and extractedwith 3-35 mL portions of pentane. The combined pentane solutions werewashed with 25 mL of water, 25 mL of brine and dried over MgSO₄.Evaporation of the pentane yielded 1.45 g of slightly yellow solid.Purification by column chromatography on silica using 5% (V/V) ethylacetate/hexane as eluent afforded 1.4 g (88%) of white solid afterrecrystallization from pentane: mp 72°-72.5° C.; ¹ H NMR delta 0.266 (s,6H), 1.022 (s, 9H), 3.958 (s, 3H), 7.19-8.53 (m, 6H); ¹³ C NMR delta-4.35, 18.23, 25.64, 52,03, 114.74, 122.87, 125.38, 125.62, 126.75,128.16, 130.87, 130.95, 137.10, 155.69, 167.36; IR (KBr) 2950, 2860,1715, 1635, 1605, 1480, 1290, 1210 cm¹ ; MS m/e (rel. intensity) 316(33, 285(7), 260 (33), 259 (100), 200 (11), 185 (13), 141 (8). ##STR28##

Methyl 6-tert-butldiphenlsilyloxy-2-naphthoate. A 10 mL round bottomflask equipped with magnetic stirrer and pressure-equalizing additionfunnel was charged with 3 mL of dry DMF, Methyl 6-hydroxy-2-naphthoate(1.01 g, 5 mmol) and tert-butyldiphenylsilyl chloride (1.51 g, 5.5mmol). The atmosphere was replaced with nitrogen and a solution ofimidazole (0.75 g, 11 mmol) in 3 mL of dry DMF was added dropwise over a15 minute period. Stirring was continued for 5 hours. The solution wasadded to 25 mL of water and extracted 3 times with 25 mL portions ofpentane. The combined pentane solutions were washed with 25 mL of brineand stored at -25° C. The crystals were collected and a second cropobtained by concentrating the mother liquor to 5 to 10 mL and cooling to-25° C. This process afforded 1.98 g (90%) of colorless crystals: mp86°-87° C.; ¹ H NMR delta 1.139 (s, 9H), 3.919 (s, 3H), 7.1-8.5 (m,16H); ¹³ C NMR delta 19.46, 26.47, 51.99, 114.62, 122.43, 125.46,126.81, 127.87, 230.07, 130.73, 130.77, 132.51, 135.46, 155.52, 167.33;IR (KBr) 3020, 2925, 2860, 1715, 1630, 1600, 1480, 1270, 1200, 690 cm⁻¹.##STR29##

(6-tert-Butyldimethylsilyloxy-2-naphthyl)methoxymethylene!adamantane(1c). A 250 mL three-neck flask was fitted with a reflux condenser. 125mL addition funnel, CaCl₂ drying tube and nitrogen line. The apparatuswas dried by means of a hot air gun and nitrogen purging. THF (150 mL)distilled from Na/benzophenone was added and the flask cooled in anice-water bath. Titanium trichloride (12 g, 78 mmol) was added rapidly(fumes in air|) followed by lithium aluminum hydride (1.425 g, 37.5mmol) in portions with vigorous stirring. The cooling bath was removedand the black mixture was allowed to warm to room temperature.Triethylamine (6 mL, 43 mmol) was added dropwise to the stirredsuspension and reflux begun. After 1 hour at reflux, a solution ofmethyl 6-tert-butyldimethylsilyloxy-2-naphthoate (2.38 g, 7.5 mmol) andadamantanone (1.15 g, 7.67 mmol) in 50 mL of dry THF was added dropwiseto the refluxing mixture over an 18 hour period. Reflux was continuedfor an additional 6 hours. The cooled reaction mixture was quenched bycareful addition of 10 mL of methanol and 10 mL of water. The mixturewas diluted with 50 mL of pentance and passed down a column of florisil(4"×1.5"), eluting with pentane, then 1:1 ether/pentane. If any of theblack material passes through the column it may be removed by extractingthe organic phase with water. The pooled organic solutions wereconcentrated on a rotary evaporator producing a yellow oil which waschromatographed on silica with 5% (V/V) ethyl acetate/hexane. Theproduct containing fractions when evaporated left 1.8 g of a yellow oilwhich afforded 1.27 g of 1c as slightly yellow crystals from coldpentane: mp 97.5°-98° C.; ¹ H NMR delta 0.250 (s, 6H), 1.024 (s, 9H),1.80-1.99 (m, 13H), 2.697 (s, 1H), 3.321 (s, 3H), 7.05-7.72 (m, 6H); ¹³C NMR delta -4.34, 18.27, 25.73, 28.39, 30.28, 32.32, 37.25, 39.13,39.28, 57.76, 114.78, 122.19, 126.32, 127.74, 128,06, 128.86, 129.44,130.88, 131.56, 134.00, 143.73, 153.70; MS m/e (rel. intensity) 435 (37,M+1), 434 (100), 377 (18), 345 (5), 188 (6), 162 (18), 14 (11), 73 (20).IR (KBr) 2940, 2915, 1630, 1600, 1480, 1265, 1250, 1090, 855, 840 cm⁻¹.##STR30##

(6-tert-Butyldiphenylsilyloxy-2-naphthyl)methoxymethylene!adamantane1d). Approximately 7 g of a 2:1 mixture of TiCl₃ and lithium aluminumhydride (Aldrich) was cautiously added to a 250 mL dry three-neck roundbottom flask containing 150 mL of dry THF maintained at 0° C. by an icebath. The resulting black mixture was stirred at 0° C. for 10 minutesand triethylamine (3.3 mL, 24 mmol) was added. The mixture was refluxedfor 1 hour and a solution of methyl tert-butyldiphenylsilylnaphthoate(1.76 g, 4 mmol) and adamantanone (600 mg, 4 mmol) in 40 mL of dry THFwas added over 6 hours. Reflux was continued for an additional 4 hoursand the mixture cooled to room temperature.

The reaction mixture was quenched by dropwise addition of 5 mL ofmethanol followed by 10 mL of water. The THF solution was decanted fromthe viscous black residue and concentrated to under 50 mL. This solutionwas diluted with ether and passed down a column of Florisil elutingfirst with pentane then with 1:1 ether/pentane. Evaporation of solventleft 1.9 g of a yellow oil. This oil was dissolved in hexane, filteredand chromatographed with 3% ethyl acetate/hexane on silica affording 900mg of a pale yellow oil which is homogeneous by TLC and NMR; ¹ H NMRdelta 1.133 (s, 9H), 1.75-2.0 (m, 13H), 2.65 (s, 1H), 3.283 (s, 3H),7.00-7.85 (m, 16H); ¹³ C NMR delta 19.49, 26.54, 28.35, 30.24, 32.29,37.23, 39.09, 57.73, 114.42, 121.67, 126.35, 127.59, 127.83, 127.94,128.61, 129.22, 129.95, 130.76, 131.51, 132.87, 133.76, 135.52, 143.67,153.55; MS m/e (rel. intensity) 558 (68), 502 (43), 501 (100), 250 (14),222 (11), 176 (19), 162 (25), 135 (11), 105 (22). ##STR31##

(6-Hydroxy-2-naphthyl)methoxymethylene!adamantane (1b). To a stirredsolution of the tert-butyldimethylsilyl protected alkene 1c (276 mg,0.635 mmol) in 10 mL of THF were added 0.65 mL of a 1.0M solution oftetra-n-butylammonium fluoride trihydrate in THF. The solution whichinstantly became bright yellow was stirred for one hour and then pouredinto a separatory funnel containing 100 mL of ether and 100 mL of water.The layers were separated and the aqueous layer extracted with another25 mL of ether. The combined ether solutions were dried with MgSO₄ andevaporated to yield an amber oil which was chromatographed on SiO₂ using15-25% ethyl acetate/hexane. There resulted 195 mg (96%) of white solid;mp 143°-4° C.; ¹ H NMR delta 1.8-2.1 (m, 13H), 2.697 (s, 1H), 3.336 (s,3H), 5.25 (s, 1H OH exchange with D₂ O), 7.08-7.76 (m, 6H); ¹³ C NMRdelta 28.37, 30.31, 32.36, 37.24, 39.12, 39.27, 57.80, 109.39, 117.89,126.06, 128.14, 128.46, 129.59, 130.48, 132.01, 134.03, 143.47, 153.66;IR (KBr) 3290, 2890, 2840, 1630, 1610, 1280, 1195, 1180, 1070, 860 cm¹ ;MS m/e (rel. intensity) 320 (100), 263 (15), 171 (50), 143 (13), 115(10). ##STR32##

6-Acetoxy-2-naphthyl)methoxymethylene!adamantane (1e). The correspondinghydroxy alkene 1b (96 mg, 0.3 mmol) was dissolved in 10 mL of CH₂ Cl₂and pyridine (244 mg, 3 mmol) under N₂. The solution was cooled in anice bath and a solution of acetyl chloride (98 mg, 1.25 mmol) in 1 mL ofCH₂ Cl₂ was added dropwise via syringe. A white precipitate formed.After two hours at 0°-5° C. TLC (SiO₂, 3:1 hexane/ethyl acetate) showedcomplete acetylation. After removal of the solvent in vacuo the solidresidue was washed with 30 mL of ether. The ether was washed with 3×25mL of water, dried over MgSO₄ and evaporated to dryness. The oilyproduct was chromatographed on silica using 4:1 hexane/ethyl acetate aseluent affording 70 mg (64%) of 1e as a white solid: ¹ H NMR delta1.8-2.1 (m, 13H), 2.347 (s, 3H), 2 (s, 1H), 3.315 (s, 3H), 7.21-7.85 (m,6H); ¹³ C NMR delta 21.08, 28.33, 30.77, 32.35, 37.19, 39.09, 39.23,57.77, 110.34, 121.28, 127.32, 128.11, 129.48, 131.15; IR (KBr) MS (70eV), m/e 362 (100), 320 (92), 263 (21), 171 (30). ##STR33##

2-tert-Butyldimethylsilyloxy-9H-fluoren-9-one. The procedure for thisreaction was the same as described above for methyl6-tert-butyldimethylsilyloxy-2-naphthoate. A solution of imidazole (0.5g, 7.4 mmol) in 2 mL of dry DMF was added to a solution of2-hydroxy-9-fluorenone (Aldrich, 0.55 g, 2.8 mmol) andtert-butyldimethylsilyl chloride (0.5 g, 3.3 mmol) in 5 ML of dry DMF togive after workup 0.74 g (84%) of a yellow oil: ¹ H NMR (CDCl₃) delta7.612-6.891 (m, 7H), 0.994 (s, 9H), 0.224 (s, 6H); ¹³ C NMR (CDCl₃ delta193.69, 157.04, 144.87, 137.52, 136.04, 134.73, 134.50, 127.92, 125.59,124.28, 121.24, 119.56, 116.22, 25.60, 18.18, -4.46. ##STR34##

2-tert-Butyldimethylsilyloxy-9H-fluoren-9-ylideneadamantane (3b). Asolution of 2tert-butyldimethylsilyloxy-9H-fluoren-9-one (0.689 g, 2.2mmol) and adamantanone (0.66 g, 4.4 mmol) in 30 mL of dry THF was addeddropwise over a period of 7 h to a refluxing mixture of TiCl₃ (6.8 g, 44mmol), LAH (0.8 g, 21 mmol) and triethylamine (3 mL) in 80 mL of dryTHF. The reaction was refluxed for an additional 12 h. The alkene wasthen isolated and purified as described above for 1a to give 0.65 g(68%) of 3b: mp 102°-105° C.; ¹ H NMR (CDCl₃) delta 7.832-6.785 (m, 7H),4.038 (s, 1H), 3.972 (s, 1H), 2.095-1.990 (m, 12H), 1.006 (s, 9H), 0.225(s, 6H); ¹³ C NMR (CDCl₃) delta 159.91, 155.06, 140.64, 139.89, 139.13,133.61, 126.29, 125.65, 124.31, 119.87, 118.71, 118.43, 116.35, 39.49,39.40, 36.90, 35.99, 35.90, 27.83, 25.81, 25.73, 18.35, -4.33. ##STR35##

2-Hydroxy-9H-fluoren-9-ylideneadamantane (3a). A solution of n-Bu₄NF.3H₂ O (1.4 mL, 1.0M) in THF was added to a stirred solution of alkene3b (0.525 g) in 10 mL of THF. The workup procedure was the same asdescribed above for 1b. The yield of 3a was 0.27 g (71%): ¹ H NMR(CDCl₃) δ7.838-6.760 (m, 7H), 4.878 (s, 1H, OH), 4.043 (s, 1H), 3.975(s, 1H), 2.079-1.977 (m, 12H); ¹³ C NMR (CDCl₃) δ154.84, 140.96, 139.68,138.97, 133.33, 126.29, 125.67, 124.34, 120.09, 118.61, 113.61, 111.73,39.45, 36.78, 35.90, 35.79, 27.72. ##STR36##

3-Hydroxy-9H-xanthen-9-one. Resorcinol (5.5 g, 50 mmol) and methylsalicylate (11.0 g, 72 mmol) were refluxed for 5 h using a Dean-Starktrap to remove H₂ O and MeOH. The resulting black oil waschromatographed over silica with 20% ethyl acetate-hexane as eluent. Ayellow solid was isolated which was subsequently recrystallized fromethyl acetate to give 1.3 g (12.3%) of 3-hydroxy-9H-xanthen-9-one (lit.mp 242° C.). Literature references to the synthesis of this compound: R.J. Patolia and K. N. Trivedi, Indian J. Chem., 22B, 444 (1983); J. S. H.Davies, F. Scheinmann, and H. Suschitzky, J. Org. Chem., 23, 307 (1958).##STR37##

3-(tert-Butyldimethylsilyloxy)-9H-xanthen-9-one.3-Hydroxy-9H-xanthen-9-one (2.00 g, 9.4 mmol) andtert-butyldimethylsilyl chloride (1.57 g, 10.4 mmol) were dissolved in20 mL of dry DMF and stirred at room temperature. Imidazole (1.46 g,21.5 mmol) was added cautiously and the solution was stirred for 4 h.The solution was then transferred to a separatory funnel and 100 mL ofhexane was added. After washing with 3-100 mL portions of H₂ O, theorganic layer was dried with MgSO₄ and concentrated. Chromatography oversilica with 5% ethyl acetate-hexane gave 2.46 g (7.5 mmol, 80.0%) of theprotected alcohol as a white solid: mp 79°-81° C:¹ H NMR (CDCl₃) delta0.296 (s, 6H), 1.019 (s, 9H), 6.85-6.89 (m, 2H), 7.353 (ddd, 1H, J=8.0,7.0, 1.0 Hz), 7.441 (ddd, 1H, J=8.5, 1.0, 0.3 Hz), 7.680 (ddd, 1H,J=8.5, 7.0, 1.7 Hz), 8.233 (m, 1H), 8.323 (ddd, 1H, J =8.0, 1.7, 0.3Hz); ¹³ C NMR (CDCl₃ delta 176.31, 161.78, 157.75, 156.23, 134.25,128.30, 126.65, 123.75, 121.93, 117.75, 117.67, 116.46, 107.43, 25.51,18.22, -4.39. ##STR38##

3-(tert-Butyldimethylsilyloxy)-9H-xanthen-9-ylideneadamantane (5b).TiCl₃ (12.0 g, 77.8 mmol) was stirred in 100 mL of dry THF at 0° C. LAH(1.56 g, 41.1 mmol) was added cautiously and the black solution wasrefluxed for 1 h. A solution of the silyloxy xanthone (2.16 g, 6.6 mmol)and 2-adamantanone (2.95 g, 19.7 mmol) in 50 mL of THF was added over 4h to the TiCl₃ -LAH solution. The resulting mixture was refluxed for 24h. The reaction was cooled to 0° C. and MeOH (10 mL) was added. Thesolution was diluted with H₂ O (200 mL) and extracted with 2-200 mLportions of hexane. The organic fraction was washed with H₂ O (400 mL),dried with MgSO₄, and concentrated. Chromatography over silica withhexane gave 1.52 g (3.4 mmol, 51.5%) of 5b as a white solid: mp137°-138° C.; ¹ H NMR (CDCl₃) delta 0.214 (s, 6H), 0.985 (s, 9H),1.85-2.07 (m, 12H), 3.45-3.55 (m, 2H), 6.585 (dd, 1H, J=8.4, 2.4 Hz),6.681 (d, 1H, J=2.4 Hz), 7.04-7.30 (m, 5H); ¹³ C NMR (CDCl₃) delta155.86, 154.77, 145.36, 127.77, 127.50, 127.05, 126.74, 122.50, 120,05,117.42, 116.44, 116.12, 114.57, 108.13, 39.50, 39.45, 37.10, 32.60,32.55, 27.96, 25.66, 18.18, -4.41; HRMS calcd 444.2484, found 444.2480;MS m/e (rel intensity) 444 (100), 443 (31), 387 (25), 253 (9); Anal.calcd for C₂₉ H₃₆ O₂ Si: C, 78.38; H, 8.11. Found: C, 78.70; H, 8.23.##STR39##

3Hydroxy-9H-xanthen-9-ylideneadamantane (5a). The silylated alkene 5b(1.18 g, 2.6 mmol) was dissolved in 10 ml of THF. n-Bu₄ NF.3H₂ O (0.94g, 3.0 mmol) was added and the yellow solution stirred for 30 min. Thesolution was then diluted with Et₂ O (100 mL), washed with H₂ O (200mL), and the organic layer was concentrated. Recrystallization fromethyl acetate gave 0.48 g (1.5 mmol, 57.7%) of 5a as a white solid:235°-240° C. (dec); ¹ H NMR (CDCl₃) delta 1.873 (2, 10H), 1.992 (s, 2H),3.472 (s, 1H), 3.529 (s, 1H), 6.70-6.76 (m, 2H), 6.96-7.04 (m, 2H),7.06-7.14 (m, 2H), 7.21-7.29 (m, 2H); HRMS calc. 330.1621, found330.1617; MS m/e (tel. intensity) 330 (100), 329 (43), 273 (37), 235(16), 197 (11), 142 (65). Anal. calcd for C₂₃ H₂₂ O₂ : C, 83.64; H,6.67. Found; C, 83.75; H, 6.69. ##STR40##

3-Acetoxy-9H-xanthen-9-ylideneadamantane (5c). Hydroxy alkene 5a (0.577g, 1.5 mmol) was dissolved in 20 mL of CH₂ Cl₂ with 1.25 mL (1.22 g,15.5 mmol) of pyridine. Acetyl chloride (0.6 mL, 0.662 g, 8.4 mmol) wasdissolved in 5 mL of CH₂ Cl₂ and added dropwise to the solution with 5a.A precipitate formed immediately. After stirring for 2 hours, thesolvent was removed to give a yellow-orange solid. This material wastreated with 50 mL of CH₂ Cl₂ to leave a white solid which was separatedby filtration. The CH₂ Cl₂ solution was then concentrated andchromatographed over silica with 5% ethyl acetate/hexane to give 0.502 g(77.2%) of 5c as a white solid: mp 162°-163° C.; ¹ H NMR (CDCl₃ delta1.80-2.05 (m, 12H), 2.265 (s, 3H), 3.45-3.55 (m, 2H), 6.833 (dd, 1H,J=8.38, 2.32 Hz), 6.961 (d, 1H, J=2.33 Hz), 7.072 (ddd, 1H, J=8.11,5.45, 2.08 Hz), 7.12-7.28 (m, 4H); ¹³ C NMR (CDCl₃) delta 20.96, 27.78,32.50, 36.88, 39.36, 110.08, 115.72, 116.41, 122.75, 124.38, 126.44,126.90, 127.42, 127.68, 146.81, 149.24, 154.86, 155.48, 169.18.##STR41##

3-Phosphate-9H-xanthen-9-ylideneadamantane, bis(tetraethylammonium) salt(5d). Phosphoryl chloride (72.98 mg, 0.48 mmol) was dissolved in drypyridine (3 mL) and stirred at 0° C. The hydroxy alkene 5a (66.35 mg,0.20 mmol) was dissolved in dry pyridine (5 mL) and added slowly to thephosphoryl chloride/pyridine solution. The resulting solution wasstirred at room temperature for 1 h. A 40% solution of Et₄ NOH in H₂ O(4 mL) was then added slowly after which the pH of the reaction solutionwas found to be approximately 8. The solution was extracted with CH₂ Cl₂(100 mL), the organic layer subsequently washed with 2-50 mL portions ofaqueous KCl (saturated). The organic layer was dried with anhydrousMgSO₄ and concentrated to give 5d as a yellow oil (29.11 mg, 22.3%): ¹ HNMR (CDCl₃ delta 1.007 (t, 24H, J=7.24 Hz), 1.70-2.00 (m, 12H),2.85-2.95 (m, 2H), 3.30-3.45 (m, 16H), 7.00-7.20 (m, 3H), 7.25-7.40 (m,2H), 7.65-7.75 (m, 1H), 8.55-8.70 (m, 1H). ##STR42##

Methyl 3-hydroxybenzoate. m-Hydroxybenzoic acid (10 g, 72.5 mmol) wasdissolved in 100 mL of methanol and the solution refluxed with acatalytic amount of HCl. After 24 hours tlc analysis on silica with 10%ethyl acetate/hexane revealed a trace of the starting benzoic acidremaining. The solution was cooled and concentrated to dryness. Thesolid residue was dissolved in 200 mL of ether and washed with 100 mL ofsaturated aq. NaHCO₃ and brine. Drying the solution over MgSO₄ andevaporating the solvent left a slightly yellow solid that was purifiedby recrystallization from benzene/cyclohexane to give methyl3-hydroxybenzoate as a white solid (6.74 g, 61%): 71°-73° C. ##STR43##

Methyl 3-tert-butyldimethylsiloxybenzoate. A 50 mL round-bottom flaskfitted with a magnetic stirrer and pressure-equalizing dropping funnelwas charged with 10 mL of DMF (dried by distillation from CaH₂). Methyl3-hydroxybenzoate (2.37 g, 16 mmol) and tert-butyldimethylsilyl chloride(3.05 g, 22 mmol) in 10 mL of dry DMF were added and the atmospherereplaced with nitrogen. A solution of imidazole (2.23 g, 33 mmol) in 10mL of dry DMF was added over 5 min and the stirring continued for 16hours at room temperature. TLC analysis over silica with 20% ethylacetate/hexane showed clean conversion to a new material. The reactionsolution was transferred to a separatory funnel containing 25 mL ofpentane and 25 mL of water. The pentane layer was removed and theaqueous phase extracted with 2-25 mL portions of pentane. The combinedpentane fractions were washed with 25 mL of brine and dried with MgSO₄.Evaporation of the pentane gave the silylated alcohol as a slightlyyellow oil (4.24 g, 100%). ##STR44##

(3-tert-butyldimethylsilyloxyphenyl)methoxymethylene!adamantane (7b). A500 mL three-neck flask was fitted with a reflux condenser, 125 mLaddition funnel, and nitrogen line. The apparatus was dried by means ofa hot air gun and nitrogen purging. Dry THF (200 mL) was added and theflask cooled in an ice bath. TiCL₃ (24 g, 156 mmol) was added rapidlyfollowed by LAH (2.8 g, 75 mmol) in portions with stirring. The coolingbath was removed and the black mixture was allowed to warm to roomtemperature. Triethylamine (12 mL, 86 mmol) was added to the stirredsuspension and refluxed for 1 hour. After this period a solution ofmethyl 3-tert-butyldimethylsilyloxybenzoate (4.40 g, 16.6 mmol) and2-adamantanone (3.0 g, 20.4 mmol) in 50 mL of dry THF was added dropwiseto the refluxing mixture over 5 hours. Refluxing was continued for anadditional 4 hours after which the reaction was cooled to roomtemperature and diluted with 100 mL of ether. The organic solution wasseparated and concentrated. Chromatography over silica with 1% ethylacetate/hexane gave 1.29 g (21%) of 7b as an oil: ¹ H NMR (CDCl₃) delta0.196 (s, 6H), 0.985 (s, 9H), 1.78-1.97 (m, 12H), 2.628 (s, 1H, 3.23 (s,1H), 3.29 (s, 3H), 6.75-7.20 (m, 4H); ¹³ C NMR (CDCl₃) delta -4.50,18.19, 25.67, 28.37, 30.16, 32.28, 37.25, 38.19, 39.01, 57.51, 119.29,121.08, 122.32, 128.87, 131.11, 136.84, 143.47, 155.37. ##STR45##Preparation of 1,2-Dioxetanes

Photooxygenation procedure. Typically a 5-10 mg sample of the alkene wasdissolved in 5 mL of CH₂ Cl₂ in the photooxygenation tube. Approximately40 mg of polystyrene-bound Rose Bengal (Sensitox I) was added and anoxygen bubbler connected. Oxygen was passed slowly through the solutionfor 5 minutes and the apparatus immersed in a half-silvered Dewar flaskcontaining Dry Ice/2-propanol. The sample was irradiated with either a250 W or 1000 W sodium lamp (GE LUcalox) and a UV cutoff filter whileoxygen was bubbled continuously. Progress of the reaction was monitoredby TLC. A spot for the highly stable dioxetanes could usually bedetected and had a R_(f) slightly less than that of the alkene. Theunstable dioxetanes decomposed during TLC so the reaction was judgedcomplete when the alkene was completely consumed. For the unstabledioxetanes, the sensitizer was filtered off at -78° C. by using a streamof nitrogen to push the solution through a Dry Ice-jacketed sinteredglass funnel and the solution stored at -78° C. This solution wasgenerally used directly for kinetic measurements. The stableadamantyl-substituted dioxetanes were filtered at room temperature,evaporated on a rotary evaporator and recrystallized from a suitablesolvent.

4-Methoxy-4-(2-naphthyl)spiro 1,2-dioxetane-3,2'-adamantane!(2a). Alkene1a (125 mg) was photooxygenated in 10 ml of CH₂ Cl₂ at -78° C. with a1000 W lamp using Sensitox I as sensitizer. TLC analysis (silica gel, 5%ethyl acetate/hexane) showed clean conversion to a more polar materialin 80 minutes. Filtration and removal of solvent produced a yellowishoil which crystallized from pentane at -25° C. only after 2 weeks togive 2a: mp 116° C.; ¹ H NMR delta 0.9-2.0 (m, 12H), 2.22 (s, 1H), 3.11(s, 1H), 3.242 (s, 3H), 7.0-8.3 (m, 7H); ¹³ C NMR delta 25.94, 26.07,31.60, 31.72, 32.31, 33.08, 33.23, 34.88, 36.42, 50.00, 95.60, 112.33,125.21, 126.47, 127.02, 127.63, 127.91, 128.67, 129,41, 132.13, 132.85,133.61.

4-(6-Hydroxy-2-naphthyl)-4-methoxyspiro 1,2-dioxetane-3,2'-adamantane!(2b). The corresponding alkene 1b (18.5 mg) was irradiated with the 1000W Na lamp in 4 ml of a 1:1 mixture of CH₂ Cl₂ and acetone cooled to -78°C. in the presence of 40 mg of Sensitox I. TLC using 10:1 CH₂ Cl₂ /MeOHshowed clean conversion to a new material. The sensitizer was removed byfiltration and the solvent evaporated giving 19 mg of 2b as a whitesolid: ¹ H NMR delta 0.9-2.0 (s, 12H), 2.2 (s, 1H), 3.093 (s, 1H), 3.241s, 3H), 7.1-7.9 (m, 6H); ¹³ C NMR delta 25.91, 26.03, 31.58, 31.68,32.33, 33.02, 33.22, 34.84, 36.40, 49.99, 95.77, 109.37, 118.35, 126.39,128.22, 129.74, 130.67, 134.95, 154.55.

4-(6-tert-Butyldimethylsilyloxy-2-naphthyl)-4-methoxyspiro!1,2-dioxetane-3,2'-adamantane!(2c). Alkene 1c (30 mg) was photooxygenated in 10 ml of CH₂ Cl₂ at -78°C. with a 1000 W lamp using Sensitox I as sensitizer. TLC analysis(silica gel, 5% ethyl acetate/hexane) showed clean conversion to a morepolar material in 60 minutes. Filtration and removal of solvent produced2c as an oil which crystallized from hexane at -25° C.; mp 107° C.; ¹ HNMR delta 0.268 (s, 6H), 1.030 (s, 9H), 1.4-2.0 (m, 12H), 2.2 (s, 1H),3.1 (s, 1H), 3.234 (s, 3H), 7.1-7.85 (m, 6H); ¹³ C NMR delta -4.33,18.23, 25.67, 25.93, 26.06, 31.59, 31.69, 32.31, 33.04, 33.19, 34.86,36.42, 49.94, 95.59, 112.44, 114.63, 122.58, 126.64, 128.50, 129.85,130.11, 134.93, 154.59.

4-(6-Acetoxy-2-naphthyl)-4-methoxyspiro1,2-dioxetane-3,2'-adamantane!(2e). Alkene 1e (14 mg) wasphotooxygenated in 4 ml of CH₂ Cl₂ at -78° C. with a 1000 W lamp using40 mg of Sensitox I as sensitizer. TLC analysis (silica gel, 25% ethylacetate/hexane) showed clean conversion to a more polar material in 20minutes. The sensitizer was removed by filtration and the solutiondiluted to 10.0 mL with dry methylene chloride to make a stock solutionwhose concentration was ca. 3.8×10⁻³ M. An aliquot injected into 3 mL ofo-xylene at 95° C. produced chemiluminescence which persisted forseveral hours.

Dispiro adamantane-2,3'- 1,2!dioxetane-4',9"-(2-tert-butyldimethylsilyloxy-9-fluorene)! (4b). Alkene 3b (100 mg) wasphotooxygenated in CH₂ Cl₂ (5 mL) containing 80 mg of Sensitox I for 4hours. Dioxetane 4b was subsequently purified by preparative tlc onsilica gel using 5% ethyl acetate/hexane: ¹ H NMR (CDCl₃) delta 0.233(s, 6H), 1.016 (s, 9H), 1.257-1.998 (m, 12H), 3.022 (bs, 2H),6.860-7.988 (m, 7H); ¹³ C NMR (CDCl₃) delta -4.44, -4.38, 18.27, 25.48,25.71, 31.85, 33.18, 33.36, 33.62, 33.73, 36.01, 94.42, 97.51, 119.32,120.82, 121.97, 126.05, 126.68, 130.24, 133.42, 140.17, 142.41, 155.39.

4-(3-tert-Butyldimethylsilyloxyphenyl)-4-methoxyspiro1,2-dioxetane-3,2'-adamantane! (8b). Alkene 7b (98.8 mg) wasphotooxygenated in 3 mL of CH₂ Cl₂ using Sensitox I. TLC analysis oversilica with 10% ethyl acetate/hexane showed clean conversion to a morepolar material in 40 min. Filtration and removal of the solvent produced8b as an oil: ¹ H NMR (CDCl₃) delta 0.195 (s, 6H), 0.989 (s, 9H),1.26-1.90 (m, 13H), 3.023 (s, 1H), 3.231 (s, 3H), 6.86-7.30 (m, 4H).

Dioxetanes 2d, 4a, 6a, 6b, 6c and 7a have been prepared using the aboveprocedures and have been shown to exhibit triggering properties similarto dioxetanes 2a-c and 2e.

Dispiro adamantane-2,3'- 1,2!dioxetane-4',9"-(3-phosphate-9H-xanthene!(6d). Alkene 5d (14.6 mg) was photooxygenated in 4 mL of CH₂ Cl₂ at -78°C. with a 1000 W high pressure sodium lamp using 56.3 mg of Sensitox Ias sensitizer. The solution was irradiated for 2 h to yield a stocksolution of 6d for the enzyme triggering experiments.

Chemiluminescence Kinetics Procedures

Rates of dioxetane decomposition were monitored by the decay ofchemiluminescence of aerated solutions. A cylindrical Pyrex vialequipped with magnet stir bar was filled with 3-4 mL of the reactionsolvent, sealed with a Teflon-lined screw cap and placed in thethermostatted sample block of the chemiluminescence-measuring apparatus(Black Box). Temperature control was provided by an external circulatingwater bath. Appropriate values for the instrument gain and optical slitsize were selected. When thermal equilibrium was reached (ca. 3 min.) analiquot of the dioxetane stock solution sufficient to achieve a finalconcentration not greater than 10⁴ M was added via pipette by openingthe top of the Black Box or via syringe through a light-tight rubberseptum located in the cover directly above the vial. The vial was sealedwith a Teflon-lined screw cap to prevent evaporation when hightemperatures were used. Measurement of the signal was begun by openingthe shutter. The chemiluminescent decay was generally recorded for atleast three half-lives. Calculation of the first-order rate constant, k,from the In(Intensity) vs. time data was performed by a computer programutilizing a standard least-squares treatment. The correlationcoefficient, r, was typically at least 0.999 and varied less than 5%between replicate samples. The observed rate was not measurablyconcentration dependent.

Activation Parameters for Dioxetane Decomposition

Activation parameters for decomposition of the dioxetanes werecalculated from plots of In k vs. 1/T (Arrhenius eq.) or In k/t vs. 1/T(Eyring eq.) by a standard least-squares linear regression analysis. Ina typical plot, the result of replicate runs at 5 to 10 temperaturesencompassing a 25°-50° C. temperature range were found to yield astraight line with a correlation coefficient of 0.999 or better.

The activation energy for chemiluminescence, E_(CL), was determined forseveral dioxetanes using the "temperature jump" method of Wilson andSchaap (T. Wilson and A. P. Schaap, J. Amer. Chem. Soc., 93, 4126(1971)). This method involved measuring the chemiluminescence intensityat one temperature, rapidly changing the temperature (2-3 min) underconditions of constant dioxetane concentration and measuring the newintensity. The activation energy of the light-producing step is given bythe relation:

    E.sub.CL =R In(I.sub.1 /I.sub.2)/(1/T.sub.2 -1/T.sub.1)

where R is the gas constant. This method has the advantage that it isunaffected by other non-luminescent, possibly catalytic, pathways fordioxetane decomposition which can complicate the determination by theisothermal method. Agreement between the activation energies determinedby the two methods indicates that only the "normal" unimolecular mode ofdecomposition is operative and that catalyzed destruction of thedioxetane by impurities is unimportant.

A third method which combines features of the other two methods wasperformed by measuring the constant light intensity at severaltemperatures by making a series of temperature steps. If the dioxetaneconcentration is unchanged then intensity is proportional to the rateconstant, k, and a plot of In I vs. 1/T has a slope of -E_(CL) /R.

Activation Energies for Decomposition of Dioxetanes 2 in Xylene.

    ______________________________________                                        Dioxetane E.sub.a                                                                              log A    k(sec.sup.-1) at 25° C.                                                         t1/2 at 25° C.                      ______________________________________                                        2a        29.7   13.2     3.17 × 10.sup.-9                                                                 6.9 yrs                                    2b        29.7   13.3     3.83 × 10.sup.-9                                                                 5.7 yrs                                    2c        27.0   11.7     8.72 × 10.sup.-9                                                                 2.5 yrs                                    ______________________________________                                    

The above results demonstrate the very high stability (long half-life)that the dioxetanes exhibit before triggering with the appropriatechemical agent or enzyme.

Acquisition of Chemiluminescence Spectra

Spectra of the chemiluminescence emission from dioxetane decompositionwere collected by conducting the reaction (either thermal or triggered)in a 1-cm square quartz cuvette in the sample compartment of a SpexFluorolog spectrofluorometer. The sample holder was thermostatted bymeans of an external water bath which circulated water/ethylene glycolthrough the block. A magnetic stirrer mounted below the sample holderassured constant temperature. Correction for the decay of thechemiluminescence intensity during the wavelength scan was made byaccumulating the spectrum in a ratio mode whereby the observed spectrumwas divided by the signal from an auxiliary detector (EMI 9781B) whichmeasures the total signal as a function of time. The monochromatorbandpass was typically 18 nm. For weakly emitting samples, severalidentical scans were performed and added together to improve thesignal-to-noise ratio.

When the chemiluminescence decays were measured at elevatedtemperatures, the concentration of dioxetane was corrected for thevolume expansion of the solvent. Temperature correction plots for allsolvents employed were constructed by measuring the change in absorbancewith temperature of a dilute solution of DBA at 404 nm or of1,2-ethanediol-bis-(3-dimethylaminobenzoate) at 347 nm. Plots of %(absorbance at 23° C.) vs. temperature over the range 23° C. to thehighest temperature used, usually about 90° C., were found to be linearso that the correction factor (<1) could be interpolated directly fromthe plot.

Procedures for Chemical Triggering of Dioxetanes

A solution of the dioxetane in a suitable solvent (e.g. o-xylene) wasplaced in the reaction vial as described above. The vial was placed inthe sample holder which was maintained at a temperature such thatthermal decomposition of the dioxetane was negligible. Instrumentparameters were selected as above and data collection started. Asolution of the releasing agent (e.g. base or fluoride) preferably inthe reaction solvent was injected by syringe into the rapidly stirreddioxetane solution. The volume of releasing agent added was generallyless than 5% of the total volume so that temperature fluctuation of thesample during the time course of the decay was minimal. The pseudo-firstorder decay was monitored for at least three half-lives.

1. Triggering the Chemiluminescence of Hydroxy-Substituted Dioxetaneswith Base: Potassium tert-butoxide induced decomposition of 2b.Treatment of a 10⁻⁴ M solution of dioxetane 2b in o-xylene with asolution of potassium t-butoxide in o-xylene (final concentration ofbase =0.005M) resulted in an intense blue chemiluminescence whichdecayed with a half-life of approximately 20 seconds at 25° C. Similarexperiments with 2b in methanol using KOH as the base or in o-xylenewith n-BuLi as the base also resulted in bright blue chemiluminescencewith similar decay rates. Base-induced decomposition of dioxetanes4a, 6aand 8a also produced chemiluminescence at room temperature.

2. Triggering the Chemiluminescence of Silyloxy-Substituted Dioxetaneswith Fluorde Ion: Fluoride ion induced decomposition of 2c. An aliquotof a methylene chloride stock solution of dioxetane 2c was injected into3 mL of 0.01M tetrabutylammonium fluoride in 2-methoxyethanol resultingin a final dioxetane concentration of 10⁻⁴ M. Blue chemiluminescence wasproduced which decayed according to pseudo-first order kinetics with ahalf-life of about 20 minutes at room temperature. (Dioxetanes 2d, 4b,6b, and 8b also undergo similar fluoride induced chemiluminescence.These dioxetanes also yield bright chemiluminescence in polar aproticsolvents such as acetonitrile). The corresponding decomposition of 2c at25° C. in the absence of fluoride ion exhibits a half-life of 2.5 years.A spectrum of the chemiluminescence obtained from the fluoridetriggering of 2c in 1:1 aqueous/2-methoxyethanol is shown in FIG. 1 withthe solid line. The fluorescence of the cleavage product (methyl6-hydroxy-2-naphthoate) from the dioxetane is also shown with the dashedline for comparison. These results demonstrate that it is the singletexcited state of the ester and not adamantanone which gives rise to theobserved chemiluminescence.

Enzymatic Triggering of Chemiluminescent Dioxetanes

1. Aryl Esterase

A secondary stock solution of the acetate-protected dioxetane 2e wasmade by evaporating an aliquot of the methylene chloride stockequivalent to 10 micromoles of dioxetane and dissolving in 5.0 mL of2-methoxyethanol to give a final concentration of 0.002M. This solutionwhen stored at 0° C. was stable indefinitely. Buffer solutions preparedin distilled water were 0.05M phosphate pH 7.6 and 8.0, 0.02M Tris(tris-hydroxymethylaminomethane maleate) pH 7.6, and pH 9.0phosphate/borate buffer. Aryl esterase (also called carboxylic esterhydrolase (P-5221)) from porcine liver was purchased from Sigma ChemicalCo. as a suspension of 11 mg of protein per mL in 3.2M (NH₄)₂ SO₄solution pH 8.0. Each mg of protein is equivalent to 260 Units, where 1Unit is defined as the amount which will hydrolyze 1 micromole of ethylbutyrate in 1 minute at pH 8.0, 25° C. When a 150 μL (0.3 μmol) aliquotof the acetoxy-dioxetane stock solution was added to 3.0 mL of pH 7.6Tris buffer at 25° C. in the Black Box, no chemiluminescence signal wasdetected. Injection of 1 μL of (0.26 units) of aryl esterase to thestirred solution caused a chemiluminescent signal to appear. Theintensity reached a maximum at about 3 minutes and decayed over a 30minute period. That this chemiluminescence is due only to anenzyme-catalyzed hydrolysis of the acetate ester function isdemonstrated by the following series of experiments:

1.) Repeating the experiment described above without either thedioxetane or the enzyme produced no chemiluminescence. 2.) Catalysis ofthe dioxetane decomposition by the medium in which the enzyme isconstituted was ruled out since a solution of 150 μL of dioxetane stockin 3 mL of Tris buffer containing 5 μL of 3 M (NH₄)₂ SO₄ produced nochemiluminescence at 25° C. 3.) When distilled water was substituted forthe Tris buffer, no chemiluminescence signal was observed, but on addingTris buffer to this solution light emission similar to that above wasproduced. 4.) In similar experiments where 150 μL of dioxetane stock in3 mL of Tris buffer was triggered with 1 μL of enzyme at 25, 37 and 50°C., the maximum light intensity, I_(MAX), increased with increasingtemperature while the rate of decay of light emission and time requiredto reach maximum intensity, t_(MAX), both decreased. 5.) Denaturing theenzyme by heating 1 μL in 3 mL of Tris buffer to 90° C. and cooling to25° C. resulted in no chemiluminescence when an aliquot of the dioxetanestock solution was subsequently added. Addition of untreated enzymepreparation to this solution again produced light. 6.) Addition of theknown enzyme inhibitor, sodium lauryl sulfate (SDS), to a solution of 3mL of Tris buffer, 150 μL of dioxetane stock solution and 1.5 μL ofenzyme when the light emission had reached a maximum caused anirreversible decrease in the intensity. The emission could be totallyextinguished by addition of sufficient SDS. The decrease in lightemission is not due to photophysical quenching of the excited statesince thermal decomposition in the same solvent system at elevatedtemperatures results in readily detectable chemiluminescence. 7.)Sequential injection of ten identical aliquots of the dioxetane stocksolution when light emission had stopped resulted in identicalchemiluminescence decay curves, both in I_(MAX) and time for completedecay of the signal. This experiment shows that the role of the enzymein the reaction is catalytic.

Competitive Inhibition. Competitive inhibitors are chemically similarsubstances which may reversibly or irreversibly impede an enzymaticreaction by competing with the substrate of interest for the enzymebinding site(s). If binding of the inhibitor is reversible (e.g. if itsproducts upon reaction at the enzyme do not bind irreversibly) then theenzymatic reaction of a given substrate may be temporarily slowed orstopped by addition of a competing substrate with a greater affinity(binding constant) for the enzyme. When the competing substrate isconsumed reaction of the first substrate may resume. If the enzymaticreaction of interest is a chemiluminescent reaction then competitiveinhibitors should cause a decrease in light intensity due to the slowerrate. In the limit where reaction of the inhibitor is much faster thanreaction of the chemiluminescent precursor, this effect should manifestitself as a temporary drop in light intensity until the competitor isconsumed followed by restoration of the previous light intensity.

This type of behavior explains the effect of the addition of the knownesterase substrates α-naphthyl acetate and β-naphthyl acetate. Thesesubstrates were shown by UV spectroscopy to be hydrolyzed by the enzymein seconds under the reaction conditions. A solution of 25 μL of thedioxetane stock (0.002M) in 3 mL of pH 7.6 phosphate buffer maintainedat 37° C. was treated with 5 μL of the enzyme to initiate thechemiluminescence. At the point of maximum emission 10 μn of 0.011Msolution of either α- or β-naphthyl acetate were added. A rapid decreasein light intensity was noted followed by restoration of the originalintensity within less than one minute.

Stability of Enzyme and Dioxetane to Reaction Conditions. Manydioxetanes are known to be destroyed via a non-luminescent pathway byacid catalyzed processes in protic solvents. Similarly, amines are alsoknown to cause the catalytic destruction of dioxetanes via anelectron-transfer process. The stability of the dioxetane to the aqueousbuffers used in the enzyme reactions, especially Tris buffer, was amatter of concern. A series of experiments were performed to assess thestability of the dioxetane in these buffers over the expected timecourse of a typical run. A comparison was made between the maximum lightintensity produced for a given buffer and temperature with delays of 0and 30 minutes before the enzyme was added. If the dioxetane weredecomposing in the buffer then I_(MAX) of the run where the dioxetanewas exposed to the buffer for 30 minutes would be lower provided theenzyme is not saturated. Since constant light levels were not seen inany runs it can be reasonably assumed that saturation kinetics did notapply here. In 0.05M phosphate buffer, pH 7.6 at 25° C. and 37° C. thepercent decrease in I_(MAX) due to the 30 minute delay was,respectively, 0 and 7% while in 0.02M Tris buffer, pH 7.6 at 25° C. a12% decrease was found and at 37° C. after a delay of one hour a 34%decrease occurred.

Chemiluminescence Spectra. The enzyme-catalyzed decomposition wascarried out in Tris buffer, pH 7.6 at room temperature in a standard1-cm cuvette in the sample compartment of a Spex Fluorologspectrofluorometer. Scanning the wavelength of the emission revealedthat the chemiluminescence spectrum (FIG. 2, dashed line) matchedexactly the fluorescence spectrum (solid line) of the expected cleavageproduct, methyl 6-hydroxy-2-naphthoate, in which the acetate esterprotecting group had been removed. The spontaneous chemiluminescencespectrum of the corresponding hydroxy-dioxetane under the sameconditions of buffer and pH was also identical. These findings takentogether are strong evidence that the chemiluminescence is initiated byrate-limiting hydrolysis of the acetyl group. It proved impossible toexcite the fluorescence spectrum of the cleavage product in the spentreaction mixture due to overlapping absorption and very intensefluorescence from the enzyme itself. Interestingly, no emission from theenzyme was detected during the chemiluminescent decomposition eventhough energy transfer to the enzyme from the excited cleavage productis energetically feasible. This might be explainable if the enzymebinding site is far removed from the fluorophore.

2. Acetylcholinesterase

Acetylcholinesterase, an enzyme of considerable biological significance,hydrolyzes acetylcholine to choline and acetic acid under physiologicalconditions. It was of interest to determine whether this enzyme wouldalso initiate the chemiluminescent decomposition of the acetyl-protecteddioxetane 2e by removal of the acetyl group. Acetylcholinesterase(C-3389) from human erythrocytes was purchased from Sigma Chemical Co.as a lyophilized powder containing phosphate buffer salts. Each mg ofprotein has an activity of 0.9 Units, 1 Unit being defined as the amountwhich will hydrolyze 1 micromole of acetylcholine per minute at pH 8.0,37° C. In a test run in 3 mL of 0.05M phosphate buffer, pH 8.0 at 37.0°C., injection of a 10 μaliquot of the dioxetane stock solution causedlight emission which lasted for 20 seconds. Addition of more dioxetaneduring this period generated more light. The enzymatic chemiluminescentreaction was reversibly inhibited by the native substrate acetylcholinein the same manner as was described above with esterase and naphthylacetate.

3. Alkaline Phosphatase

A cuvette containing 3 mL of a buffer solution of2-amino-2-methyl-l-propanol (Sigma Chemical Co., pH=10.3, 1.5M) wasplaced in the black box at 37° C. A portion (200 μL) of the dioxetanestock solution (6d in CH₂ Cl₂) was added to this buffer solution.Subsequent addition of 10 μL of an alkaline phosphatase suspensionSigma, Type VII-S from Bovine Intestinal Mucosa, suspension in 3.2M(NH₄)₂ SO₄ ! gave rise to chemiluminescence over a period ofapproximately 2-3 min indicating the enzymatic triggering of thedioxetane. Similar results were obtained with alkaline phosphataseobtained from an alternate biological source (Sigma, Type III fromEscherichia coli, suspension in 2.5M (NH₄)₂ SO₄, 100 units/mL).

I claim:
 1. The method for generating light which comprises:(a)providing a stable 1,2-dioxetane of the formula ##STR46## wherein ArOXrepresents an aryl group substituted with an X-oxy group which forms anunstable oxide intermediate 1,2-dioxetane compound when triggered toremove X by an activating agent so that the unstable 1,2-dioxetanecompound decomposes to form light and two carbonyl containing compoundsof the formula ##STR47## wherein X is a chemically labile group which isremoved by the activating agent to form the unstable oxide intermediate1,2-dioxetane and wherein A are passive organic groups which allow thelight to be produced; and (b) decomposing the stable 1,2-dioxetane withthe activating agent.
 2. The method for generating light whichcomprises:(a) providing a stable 1,2-dioxetane compound of the formula##STR48## wherein R₁ and R₂ together and R₃ and R₄ together can bejoined as spirofused alkylene and aryl rings, wherein at least one of R₁and R₂ or R₃ and R₄ is an aryl group substituted with an X-oxy groupwhich forms an unstable oxide intermediate 1,2 dioxetane compound whentriggered to remove X by an activating agent selected from acids, bases,salts, enzymes, inorganic and organic catalysts and electron donors sothat the unstable 1,2-dioxetane compound decomposes to form light andtwo carbonyl containing compounds of the formula: ##STR49## whereinthose of R₁, R₂, R₃ or R₄ which are unsubstituted by an X-oxy group arecarbon containing organic groups which provide stability for the stable1,2-dioxetane compound and wherein X is a chemically labile group whichis removed by the activating agent to form the unstable oxideintermediate; and (b) decomposing the stable 1,2-dioxetane with theactivating agent.
 3. The method for generating light which comprises:(a)providing a stable dioxetane compound of the formula: ##STR50## whereinR₁ is selected from alkyl, alkoxy, aryloxy dialkylamino, trialkyl oraryl silyloxy and aryl groups including spirofused aryl groups with R₂,wherein R₂ is an aryl group which can include R₁ and is substituted withan X-oxy group which forms an unstable oxide intermediate 1,2-dioxetanecompound when triggered to remove X by an activating agent selected fromacids, bases, salts, enzymes, inorganic and organic catalysts andelectron donors so that the unstable 1,2-dioxetane compound decomposesto form light and two carbonyl containing compounds of the formula:##STR51## wherein X is a chemically labile group which is removed by theactivating agent to form the unstable oxide intermediate and wherein R₃and R₄ are selected from aryl and alkyl groups which can be joinedtogether as spirofused polycyclic alkyl and polycyclic aryl groups; and(b) decomposing the stable 1,2-dioxetane with the activating agent. 4.The method of claim 1 wherein the 1,2-dioxetane compound is decomposedby an enzyme from a biological source which removes X.
 5. The method ofclaim 4 wherein the 1,2-dioxetane has a phosphate group as the X-oxygroup and the enzyme is alkaline phosphatase.
 6. The method of claim 4wherein the 1,2-dioxetane has an acetoxy group as the OX group and theenzyme is acetylcholinesterase.
 7. The method of claim 4 wherein the1,2-dioxetane has an acetoxy group as the OX group and wherein theenzyme is arylesterase.
 8. The method of claim 8 wherein the method isperformed as part of an immunoassay.
 9. The method of claim 2 wherein R₃and R₄ are combined together to form a polycyclic polyalkylene groupcontaining 6 to 30 carbon atoms.
 10. The method of claim 9 wherein R₂ isselected from naphthyl and phenyl groups containing the X-oxy group andwherein R₁ is a methoxy group.
 11. The method of claim 10 wherein theX-oxy group is selected from hydroxyl, trialkyl or aryl silyloxy,inorganic oxyacid salt, oxygen-pyranoside, aryl carboxyl esters andalkylcarboxyl esters and wherein X is removed by the activating agent.12. The method of claim 1 wherein the X-oxy group is a phosphate groupand wherein the phosphate group is removed with a phosphatase as theactivating agent.
 13. The method of claim 11 wherein the X-oxy group isselected from alkyl and aryl carboxyl ester groups and wherein thecarboxyl group is removed with an esterase.
 14. The method of claim 2wherein R₃ and R₄ are combined together to form a polycyclicpolyalkylene group containing 6 to 30 carbon atoms and wherein R₁ and R₂are combined together as a spirofused aryl group which is substitutedwith the X-oxy group.
 15. The method of claim 14 wherein the spirofusedaryl group is a 9H-xantheny-9-yl group.
 16. The method of claim 15wherein the spirofused aryl group is a 9H-fluoren-9-yl group.
 17. Themethod of claim 15 wherein the 9H-xanthen-9-yl is substituted with analkyl carboxyl ester group as the OX group and wherein the alkylcarboxyl group is removed by an esterase.
 18. The method of claim 15wherein the 9H-xanthenyl-9-yl group is substituted with a PO₄ group asthe OX group and wherein a PO₃ group is removed from the PO₄ group by aphosphatase.
 19. The method of claim 2 wherein the 1,2-dioxetanecompound is decomposed by an enzyme from a biological source whichremoves X.
 20. The method of claim 19 wherein the 1,2-dioxetane has aphosphate group as the X-oxy group and the enzyme is alkalinephosphatase.
 21. The method of claim 19 wherein the 1,2-dioxetane has anacetoxy group as the X-oxy group and the enzyme is acetylcholinesterase.22. The method of claim 13 wherein the 1,2-dioxetane has an acetoxygroup as the X-oxy group and wherein the enzyme is arylesterase.
 23. Themethod of claim 2 wherein the method is performed as part of animmunoassay.
 24. A method for generating light in a chemiluminescentassay using an enzyme from a biological source which comprises:(a)providing a compound of the formula: ##STR52## wherein R₁ and R₂ takentogether are a spirofused polycyclic aryl group having an aryl ringsubstituted with an X-oxy group wherein X is removeable by enzymatictriggering ahd R₃ and R₄ taken together are an adamantyl group; and (b)triggering the compound to generate light using an enzyme from abiological source.
 25. The method of claim 24 wherein the spirofusedpolycyclic aryl group is xanthenyl with an aryl ring substituted withthe X-oxy group.
 26. The method of claim 25 wherein the X-oxy group is aphosphate.
 27. A method for generating light in a chemiluminescent assayusing an enzyme from a biological source which comprises:(a) providing acompound of the formula: ##STR53## wherein R₁ is methoxy, R₂ is an arylgroup substituted with an X-oxy group wherein X is removable byenzymatic triggering and R₃ and R₄ taken together are an adamantylgroup; and (b) triggering the compound to generate light using an enzymefrom a biological source.
 28. In an immunoassay method wherein there isan optically detectable reaction, the improvement wherein said opticallydetectable reaction includes the reaction, with an enzyme, of adioxetane having the formula ##STR54## where T is an adamantyl group; Yis naphthyl; X is methoxy; and Z is an enzyme-cleavable group,so thatsaid enzyme cleaves said enzyme-clearable group from said dioxetane toform a negatively charged substituent bonded to said dioxetane, saidnegatively charged substituent causing said dioxetane to decompose toform a luminescent substance comprising said group Y of said dioxetane.29. The method of claim 28 wherein Z is acetoxy.